Although the gene responsible for CF was discovered more than 2 decades ago, it still remains unclear how CFTR defects lead to the airway disease that is responsible for most CF deaths. Such slow progress can be attributed to the lack of an adequate animal model of CF airway disease. Although CF mice have illuminated so many aspects of CF pathophysiology, they do not display a human-like CF airways disease. The need for different animal models of CF prompted the creation of CF pig and ferret models. We believe this is the first report of early phenotypic features in a genetically engineered ferret model of CF, demonstrating that it shares many of the abnormalities seen in newborn humans with CF. To our knowledge, CF is the first human disease for which directed engineering has generated 2 non-rodent knockout models, and comparative studies on organ-specific CF disease phenotypes in 4 species (human, mouse, ferret, and pig) will greatly expand our understanding of CF pathogenesis.
The greatest cause of CF neonatal mortality in all species is intestinal obstruction, but with important differences in penetrance, age at onset, and phenotype. Human with CF and CF pigs and ferrets display a true MI at birth, whereas CF mice develop intestinal complications at a different stage in development (i.e., weaning to solid chow). The penetrance of MI varies greatly among humans with CF (~15%), CF ferrets (~75%), and CF pigs (100%), and a wide variance in intestinal obstruction is also observed in CF mice (0%–100%) depending on the strain background (5
). Modifier genes appear to influence the occurrence of MI in infants with CF based on monozygous and dizygous CF twin/triplet studies (33
), and our studies also suggest that the hob exerts a genetic influence for development of MI in CFTR–/–
ferrets. Complete penetrance of MI in the CFTR–/–
piglets studied to date may be a consequence of their intestinal anatomy and/or their inbred status.
The severity of CF pancreatic pathology at birth differs widely across the species, with pigs being most severe (7
), ferrets and humans being similar (16
), and mice being least severe (6
). The sparing of the pancreas in newborn mice has been attributed to alternative Ca+
-activated chloride channels in this organ (34
), and it will be interesting to determine whether this single factor can account for the variation across all 4 species. The vas deferens is another tissue that demonstrates variable CF disease pathology between the species. Congenital bilateral absence of the vas deferens is diagnosed in nearly all (~99%) of adult males with CF (18
). Even though “congenital absence” implies that this structure is not present at birth, most infant males with CF have an intact vas deferens at birth (17
), and increased detection rate in younger patients suggests that disease in this tissue is a degenerative process that may begin prior to birth in some individuals and culminate with destruction by adulthood (19
). Interestingly, with the exception of the CF ferret that demonstrates an absent or degenerate vas deferens at birth, other CF animal models have not replicated this phenomenon — CF mice have minor histologic changes to the vas deferens but are fertile (35
), and CF pigs appear to have intact vas deferens at birth (7
). Whether CF pigs will develop disease of the vas deferens in adulthood remains to be determined.
Of all species, CF newborn ferrets have the most severely impaired nutritional status at birth. The ferret intestinal tract is unique in comparison with those of humans, pigs, and mice. As an obligate carnivore, ferrets lack a cecum, which is found in humans, pigs, and mice; this structure is known to assist in the digestion of plant material. The ferret also has a shorter intestinal transit time in comparison to other species (36
). These features likely impose special nutrient requirements, which to date remain to be defined at the molecular level. The complete lack of weight gain in newborn CF ferrets suggests that intestinal CFTR plays a critical role in nutritional absorption in this species. Additionally, the pancreatic pathology and bile acid–dependent liver abnormalities observed at birth in CF ferrets may also play a role in their poor nutrition.
Oral administration of a proton-pump inhibitor significantly improved nutrition in CF ferrets, suggesting that CFTR-dependent perturbations in GI pH may have a significant impact on the absorption of fat and/or other key nutrients in this species. Proton-pump inhibitors have been extensively used in CF patients suffering from sustained steatorrhea while on pancreatic enzymes, and these drugs appear to improve nutritional status according to some reports (29
). Furthermore, reduced GI pH in a Cftr–/–
mouse model has been suggested to impair lipolysis and fat absorption by the intestine (wild type, 94% ± 0.3% absorption vs. knockout, 89.7% ± 1.2% absorption), despite normal lipase and bicarbonate secretion by the pancreas (39
), and this defect was corrected by oral administration of a proton-pump inhibitor. Interestingly, elevated bile salt secretion into the feces was also observed in Cftr–/–
mice, despite normal levels of bile salt secretion by the biliary system (39
). The authors of this study concluded that Cftr–/–
mice suffer from fat malabsorption due to impairment of the duodenal bicarbonate production that is required for efficient lipolysis and uptake of fatty acids (39
). It is interesting, however, that ΔF508-CFTR mice do not suffer from the same lipolysis and fatty acid uptake defects seen in Cftr–/–
). Although further studies on intestinal biology in the ferret are needed to understand how CFTR in this organ might directly influence lipid absorption in this species, it seems plausible that CFTR–/–
ferrets suffer from a relatively pronounced intestinal pH imbalance that influences fat absorption in a similar manner as in Cftr–/–
mice. These and other issues will likely be clarified by the production of a gut-corrected CFTR–/–
Understanding how CFTR malfunction contributes to the extreme GI phenotypes in these CF animal models should help to improve management of the more subtle pathologies seen in humans with CF. However, a major impetus to controlling or genetically eliminating the GI pathology is to allow the models to grow to maturity so that the natural progression of lung disease can be determined. Thus far, we know that the basic defects in airway bioelectric and submucosal gland secretory properties reproduce those seen in human CF. Furthermore, rearing CFTR–/–
kits under protocols that improved nutrition did not completely prevent early fatal lung infections that were also seen in nutritional compromised animals. Despite improved nutrition, neonatal CFTR–/–
kits had increased bacterial counts and isolates from BAL fluid that subsided after the first week. After the first week, lung infections did not appear to be the primary cause of death in CFTR–/–
kits. It is interesting to speculate that neonatal aspiration events may serve as inoculation mechanisms, and defective bacterial eradication compromises the ability of the CF lung to clear normal early pathogens like control animals. Although the number of animals analyzed remains low, our observations of early fatal lung infections and rectal prolapse in CFTR–/–
kits is also similar to clinical observations in CF infants during the early 1950s (40
). In this case study of 68 CF infants, over 60% died before the age of 1 year, while only 15% survived 2–13 years. More than half of the CF infants who contracted lung infection at birth died (excluding those with MI), and failure to thrive during the neonatal period despite a ravenous appetite was common. These observations are quite similar to our findings with CFTR–/–
kits. As with humans, the improved use of broad-spectrum antibiotics during the neonatal period may help to improve survival in this ferret model of CF. These initial findings in the lung and other organs of CFTR–/–
ferrets, suggest that the ferret may be a useful model for dissecting CF pathophysiology and developing therapies. This report also describes the production of what we believe to be the first transgenic ferret using SCNT, and such methods will expand opportunities for genetically dissecting CF pathogenesis in the ferret model.